Lateral Packing Density Research Articles

Unravelling the orientation of the inositol-biphosphate ring and its dependence on phosphatidylinositol 4,5-bisphosphate cluster formation in model membranes

HypothesisInositol phospholipids are well known to form clusters in the cytoplasmic leaflet of the plasma membrane that are responsible for the interaction and recruitment of proteins involved in key biological processes like endocytosis, ion channel activation and secondary messenger production. Although their phosphorylated inositol ring headgroup plays an important role in protein binding, its orientation with respect to the plane of the membrane and its lateral packing density has not been previously described experimentally. ExperimentsHere, we study phosphatidylinositol 4,5-bisphosphate (PIP2) planar model membranes in the form of Langmuir monolayers by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry to elucidate the relation between lateral (in-plane) and perpendicular (out-of-plane) molecular organization of PIP2. FindingsDifferent surface areas were explored through monolayer compression, allowing us to correlate the formation of transient PIP2 clusters with the change in orientation of the inositol-biphosphate headgroup, which was experimentally determined by neutron reflectometry.

Fluorescence Studies of Nicotinic Acetylcholine Receptor and Its Associated Lipid Milieu: The Influence of Erwin London's Methodological Approaches.

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

P-glycoprotein (ABCB1) - weak dipolar interactions provide the key to understanding allocrite recognition, binding, and transport.

P-glycoprotein (ABCB1) is the first discovered mammalian member of the large family of ATP binding cassette (ABC) transporters. It facilitates the movement of compounds (called allocrites) across membranes, using the energy of ATP binding and hydrolysis. Here, we review the thermodynamics of allocrite binding and the kinetics of ATP hydrolysis by ABCB1. In combination with our previous molecular dynamics simulations, these data lead to a new model for allocrite transport by ABCB1. In contrast to previous models, we take into account that the transporter was evolutionarily optimized to operate within a membrane, which dictates the nature of interactions. Hydrophobic interactions drive lipid-water partitioning of allocrites, the transport process's first step. Weak dipolar interactions (including hydrogen bonding, π-π stacking, and π-cation interactions) drive allocrite recognition, binding, and transport by ABCB1 within the membrane. Increasing the lateral membrane packing density reduces allocrite partitioning but enhances dipolar interactions between allocrites and ABCB1. Allocrite flopping (or reorientation of the polar part towards the extracellular aqueous phase) occurs after hydrolysis of one ATP molecule and opening of ABCB1 at the extracellular side. Rebinding of ATP re-closes the transporter at the extracellular side and expels the potentially remaining allocrite into the membrane. The high sensitivity of the steady-state ATP hydrolysis rate to the nature and number of dipolar interactions, as well as to the dielectric constant of the membrane, points to a flopping process, which occurs to a large extent at the membrane-transporter interface. The proposed unidirectional ABCB1 transport cycle, driven by weak dipolar interactions, is consistent with membrane biophysics.

Non-ionic Surfactants as a P-Glycoprotein(P-gp) Efflux Inhibitor for Optimal Drug Delivery-A Concise Outlook.

Significant research efforts have been devoted to unraveling the mystery of P-glycoprotein(P-gp) in drug delivery applications. The efflux membrane transporter P-gp is widely distributed in the body and accountable for restricting drug absorption and bioavailability. For these reasons, it is the primary cause of developing multidrug resistance (MDR) in most drug delivery applications. Therefore, P-gp inhibitors must be explored to address MDR and the low bioavailability of therapeutic substrates. Several experimental models in kinetics and dynamic studies identified the sensitivity of drug molecules and excipients as a P-gp inhibitor. In this review, we aimed to emphasize nonionic surface-active agents for effective reversal of P-gp inhibition. As it is inert, non-toxic, noncharged, and quickly reaching the cytosolic lipid membrane (the point of contact with P-gp efflux protein) enables it to be more efficient as P-gp inhibitors. Moreover, nonionic surfactant improves drug absorption and bioavailability through the various mechanism, involving (i) association of drug with surfactant improves solubilization, facilitating its cell penetration and absorption; (ii) weakening the lateral membrane packing density, facilitating the passive drug influx; and (iii) inhibition of the ATP binding cassette of transporter P-glycoprotein. The application of nonionic surfactant as P-gp inhibitors is well established and supported by various experiments. Altogether, herein, we have primarily focused on various nonionic surfactants and their development strategies to conquer the MDR-causing effects of P-gp efflux protein in drug delivery. Graphical Abstract.

Interfacial Polarization and Ionic Structure at the Ionic Liquid-Metal Interface Studied by Vibrational Spectroscopy and Molecular Dynamics Simulations.

Ionic liquids (ILs) have both fundamental and practical value in interfacial science and electrochemistry. However, understanding their behavior near a surface is challenging because of strong Coulomb interactions and large and irregular ionic sizes, which affect both their structure and energetics. To understand this problem, we present a combined experimental and computational study using a vibrational probe molecule, 4-mercaptobenzonitrile, inserted at the junction between a metal and a variety of ILs. The vibrational frequency of the nitrile in the probe molecule reports on the local solvation environment and the electrostatic field at this junction. Within the ethylmethyl imidazolium (EMIM+) cation family of ILs, we varied the anions over a range of sizes and types. Complementing our surface spectroscopy, we also ran molecular dynamics simulations of these interfaces to better understand the ionic structures that produced the measured fields. The magnitude of the frequency shifts, and thereby fields, shows a general correlation with the size of anions, with larger anions corresponding to smaller fields. We find that the source of this correlation is partial intercalation of smaller anions into the probe monolayer, resulting in tighter packing of ionic layers near the surface. Larger anions reduce the overall lateral ion packing density near the surface, which reduces the net charge per unit area and explains the smaller observed fields. The insight from this work is important for developing a fundamental picture of concentrated electrolytes near interfaces and can help with designing ILs to create tailored electric fields near an electrode.

Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·104 and 2.82·104 M−1, respectively) and lower for DOTAP (0.46·104 M−1). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.

Soft Polymer Janus Nanoparticles at Liquid–Liquid Interfaces

AbstractSoft polymeric Janus nanoparticles (JNPs), made from polystyrene‐b‐poly(butadiene)‐b‐poly(methylmethacrylate), PS‐PB‐PMMA, triblock terpolymers, assemble into a monolayer at the water–oil interface to reduce interfacial tension. The extent to which the polymer chains can deform influences the packing density of the JNPs at the interface. The longer the polymer chains are relative to the core, the softer are the JNPs, resulting in a JNPs assembly with a lower initial lateral packing density. The interfacial activity of JNPs can be further tuned by complexation of the PMMA chains with lithium ions that are introduced into the water phase. This work provides a fundamental understanding of soft JNPs packing at the water–oil interface and provides a strategy to tailor the areal density of soft JNPs at liquid–liquid interface, enabling the design of smart responsive structured‐liquid systems.

Soft Polymer Janus Nanoparticles at Liquid–Liquid Interfaces

Soft polymeric Janus nanoparticles (JNPs), made from polystyrene-b-poly(butadiene)-b-poly(methylmethacrylate), PS-PBPMMA, triblock terpolymers, assemble into a monolayer at the water/oil interface to reduce interfacial tension. The extent to which the polymer chains can deform, influences the packing density of the JNPs at the interface. The longer the polymer chains are relative to the core, the softer are the JNPs, resulting in a JNPs assembly with a lower initial lateral packing density. The interfacial activity of JNPs can be further tuned by complexation of the PMMA chains with lithium ions that are introduced into the water phase. This work provides a fundamental understanding of soft JNPs packing at the water/oil interface and provides a strategy to tailor the areal density of soft JNPs at liquid/liquid interface, enabling the design of smart responsivestructured-liquid systems.

CoQ10 selective miscibility and penetration into lipid monolayers with lower lateral packing density

CoQ10 is ubiquitously present in eukaryotic cells. It acts as electron carrier in the electron transport chain of the inner membrane of the mitochondria to facilitate aerobic cellular respiration. A highly stable lipid nanodispersion formulation containing CoQ10 (BPM31510) is currently in clinical investigation for treatment of cancer. This study was designed to determine whether biophysical interactions between CoQ10 and lipid, in part, explain the observed stability and cellular accumulation of CoQ10 in cells and tissues. A lipid monolayer at the air-water interface was used as an experimental membrane model to measure CoQ10 penetration and solubility. Lipid monolayers with varying proportions of CoQ10 were laterally compressed to measure CoQ10 miscibility and lateral organization. Additionally, lipid monolayers with varying lateral packing densities were spread at the air-water interface and CoQ10 was injected in proximity to measure its rate of penetration. Our results demonstrate that CoQ10 selectively penetrates into lipid monolayers with a lower lateral packing density, and is excluded by monolayers of higher packing densities. Data also indicates that CoQ10-lipid mixing is non-ideal. CoQ10 presence in lipid monolayers is biphasic, with one phase occupying the interstitial space between the DMPC lipids, and the other phase is present as pure CoQ10 domains. This work provides further insight into mechanism of action of CoQ10 based formulations that can significantly increase intracellular CoQ10 concentration to show pleotropic effects on cellular functions.

Surface force and vibrational spectroscopic analyses of interfacial water molecules in the vicinity of methoxy-tri(ethylene glycol)-terminated monolayers: mechanisms underlying the effect of lateral packing density on bioinertness

Unequivocal dependence of bioinertness of self-assembled monolayers of methoxy-tri(ethylene glycol)-terminated alkanethiol (EG3-OMe SAMs) on their packing density has been a mystery for more than two decades. We tackled this long-standing question by performing surface force and surface-enhanced infrared absorption (SEIRA) spectroscopic measurements. Our surface force measurements revealed a physical barrier of interfacial water in the vicinity of the Au-supported EG3-OMe SAM (low packing density), whereas the Ag-supported one (high packing density) did not possess such interfacial water. In addition, the results of SEIRA measurements clearly exhibited that hydrogen bonding states of the interfacial water differ depending on the substrates. We also characterized the bioinertness of these SAMs by protein adsorption tests and adhesion assays of platelet and human umbilical vein endothelial cells. The hydrogen bonding states of the interfacial water and water-induced interaction clearly correlated with the bioinertness of the SAMs, suggesting that the interfacial water plays an important role determining the interaction of the SAMs with biomolecules and cells.

Relative Contribution of Lateral Packing Density to Albumin Adsorption on Monolayers.

The effect of functional group density on protein adsorption is systematically studied to support ongoing efforts in molecular imprinting of surfaces and bulk materials. In these applications, functional commodity chemicals are molded to complement the shape and chemistry of the target molecule. Here, we study the relationship between bovine serum albumin adsorption and ligand density for carboxylate, alcohol, and alkyl terminal groups. Control surfaces consisting of densely packed self-assembled monolayers (SAMs) are contrasted with low-density SAMs formed through thiol-yne chemistry. Direct comparison consistently yielded greater protein adsorption on low-density SAMs than conventional pure component SAMs of the same functional group. Critically, the carboxylate and alcohol low-density SAMS are more hydrophobic than their analogous dense SAMs. Mixed functional group, dense SAMs were formed with alkyl diluents to match the hydrophobicity of the low-density SAMs. Once hydrophobicity is matched, the dense carboxylate and alcohol SAMs have higher adsorption than the low-density SAMs. We conclude (1) surface charge and hydrophobicity trends dominate over surface density contributions; (2) when hydrophobicity is matched, greater adsorption occurs on dense hydrophilic groups than on lower density hydrophilic groups; (3) when hydrophobicity is matched, greater adsorption occurs on lower density hydrophobic groups than on higher density hydrophobic groups.

Preferential Delivery of BPM 31510 into Tumorigenic Cells Based on Biophysical Interactions

Preferential drug delivery to cancer cells has remained elusive. Current approaches utilize ligand-receptor interactions or enhance vasculature in tumor area, but do not take into consideration the significant difference in organization and dynamics of tumorigenic cells surface compared to non- tumorigenic counterpart. It is extensively reported that tumorigenic cells have lower lateral packing density that also relates to their metastatic and invasive potential. Herein, we report a preliminary investigation on BPM 31510, a CoQ10 based lipid nanodispersion that demonstrate preferential incorporation into membranes with lower lateral packing density. We first employed lipid membranes at air-water interface with lower and higher packing densities as a mimic of tumorigenic and non-tumorigenic cell surfaces and injected BPM 31510 in aqueous sub-phase. We observed a decrease in BPM 31510 incorporation with an increase in lateral packing density of the membranes. Later, intracellular concentration of CoQ10 following treatment with BPM 31510 was compared in nine tumorigenic cells lines and five non-tumorigenic cell lines. The uptake of BPM 31510 was much higher in tumorigenic cells compared to non-tumorigenic cells. Data further suggest suggests that free energy related to addition of BPM 31510 into the cell surface is significantly lower in tumorigenic cells compared to non-tumorigenic counterpart. Overall, this work employ BPM 31510 as model system to demonstrate how preferential drug delivery into tumorigenic cell surface can be achieved by utilizing its lower lateral packing density compared to non-tumorigenic counterpart.

Confinement and Integration Density of Plasmonic Waveguides

In general, plasmonic waveguides exhibit a tradeoff between propagation loss and mode confinement—the smaller the mode size, the higher the propagation loss. We compare the confinement and loss of three commonly used plasmonic waveguides. We define two distinct figures of merit based on application for which the plasmonic waveguide is designed: 1) applications which require high power density and 2) applications which require high lateral packing density. The first figure of merit is based on the power density and propagation loss, whereas the second depends on the coupling length and the propagation loss. We have identified the relative advantages and limitations of the plasmonic waveguides for these different applications.

Binding behaviors of protein on spatially controlled poly[oligo(ethylene glycol) methacrylate] brushes grafted from mixed self-assembled monolayers on gold

Binding behaviors of streptavidin were investigated with different lateral packing densities of biotin-functionalized, non-biofouling pOEGMA brushes, synthesized by surface-initiated polymerization from mixed SAMs with different mole fractions of the polymerization initiator on gold surfaces.

High performance Langmuir–Schaeffer film transistors based on air stable n-type diperylene bisimide

Diperylene Bisimide (DIPP–diPBI) mono- and/or multi-layer film using Langmuir–Schaeffer (LS) techniques has been fabricated and the OFET device performance based on the as-prepared LS film is investigated. The thickness of monolayer film is determined to be 2.3nm by using atomic force microscopy, which is closely matched with the interplanar spacing estimated from the XRD spectra. The length of molecular long axis is measured to be 21.9Å from the DFT optimized configuration, indicating that the long axis of molecule in LS film approximately stands upright on hydrophobic substrates. The absorption maximum at 417nm shows a good linear corrleation with the layer number, proving the obtained films are deposited in a layer-by-layer fassion. The film with precision control of the long-range order and lateral packing density by LS deposition exhibits good electron injection properties and high FET device performance. The charge transport behavior is also investigated as a function of the layer number of LS film. The electron mobility gradually increases with the number of layers and saturates at a plateau with a mean value of 0.03cm2V−1s−1 in the atmosphere upon completion of the first eight layers. It is a direct evidence of physical size of charge transport layer. Furthermore, the fabricated FET device exhibits long-time stability in the air. The integration of LS method with air stability of the n-type compound affords an opportunity to explore solution-phase self-assembly and electronic devices fabrication with controllable molecular layers.

Crystallization of Poly(γ-benzyll-glutamate) in Thin Film Solutions: Structure and Pattern Formation

We have investigated the formation of poly(γ- benzyl L-glutamate) (PBLG) crystals from semidilute thin film solutions. Crystallization was initiated by adding methanol, a nonsolvent. After drying, each PBLG crystal possessed an internal domain structure exhibiting a zigzag pattern consisting of parallel stripes of alternating orientations between domains. X-ray scattering and electron diffraction revealed within these crystals a pseudohexagonal packing of the PBLG α-helices with their axis oriented parallel to the plane of the substrate. On the basis of optical anisotropy studies, it could be shown that the orientation of the helix axis was parallel to the stripes. While forming in solution, the objects are assumed to consist of a hexagonal columnar liquid crystalline phase. Upon drying, lateral packing density of the helices increased and resulted in a net dilative strain perpendicular to the columns, which is supposed to cause the formation of zigzag patterns.

Oxidized Phosphatidylcholines Promote Phase Separation of Cholesterol-Sphingomyelin Domains

Lipid lateral segregation in the plasma membrane is believed to play an important role in cell physiology. Sphingomyelin (SM) and cholesterol (Chol)-enriched microdomains have been proposed as liquid-ordered phase platforms that serve to localize signaling complexes and modulate the intrinsic activities of the associated proteins. We modeled plasma membrane domain organization using Langmuir monolayers of ternary POPC/SM/Chol as well as DMPC/SM/Chol mixtures, which exhibit a surface-pressure-dependent miscibility transition of the coexisting liquid-ordered and -disordered phases. Using Brewster angle microscopy and Langmuir monolayer compression isotherms, we show that the presence of an oxidatively modified phosphatidylcholine, 1-palmitoyl-2-azelaoyl-sn-glydecero-3-phosphocholine, efficiently opposes the miscibility transition and stabilizes micron-sized domain separation at lipid lateral packing densities corresponding to the equilibrium lateral pressure of ∼32 mN/m that is suggested to prevail in bilayer membranes. This effect is ascribed to augmented hydrophobic mismatch induced by the oxidatively truncated phosphatidylcholine. To our knowledge, our results represent the first quantitative estimate of the relevant level of phospholipid oxidation that can potentially induce changes in cell membrane organization and its associated functions.

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.

Systematic approach to electrostatically induced 2D crystallization of nanoparticles at liquid interfaces

We report an experimental demonstration of a strategy for inducing two-dimensional (2D) crystallization of charged nanoparticles on oppositely charged fluid interfaces. This strategy aims to maximize the interfacial adsorption of nanoparticles, and hence their lateral packing density, by utilizing a combination of weakly charged particles and a high surface charge density on the planar interface. In order to test this approach, we investigated the assembly of cowpea mosaic virus (CPMV) on positively charged lipid monolayers at the aqueous solution surface, by means of in situX-ray scattering measurements at the liquid–vapor interface. The assembly was studied as a function of the solution pH, which was used to vary the charge on CPMV, and of the mole fraction of the cationic lipid in the binary lipid monolayer, which set the interface charge density. The 2D crystallization of CPMV occurred in a narrow pH range just above the particle's isoelectric point, where the particle charge was weakly negative, and only when the cationic-lipid fraction in the monolayer exceeded a threshold. The observed 2D crystals exhibited nearly the same packing density as the densest lattice plane within the known 3D crystals of CPMV. The above electrostatic approach of maximizing interfacial adsorption may provide an efficient route to the crystallization of nanoparticles at aqueous interfaces.

Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.